Xylanases are a group of enzymes with wide commercial utility. Major applications of xylanases include pulp biobleaching in the production of paper, clarifying agents in juices and wines, as a supplement to improve digestibility of poultry and swine feed and as a washing agent of precision devices and semiconductors (e.g. U.S. Pat. No. 5,078,802).
In the manufacturing of pulp for the production of paper, fibrous material is subjected to high temperatures and pressures in the presence of chemicals. This treatment converts the fibers to pulp and is known as pulping. Following pulping, the pulp is bleached. Xylanase enzymes are used to enhance the bleaching of the pulp. The xylanase treatment allows subsequent bleaching chemicals such as chlorine, chlorine dioxide, hydrogen peroxide, or combinations of these chemicals, to bleach pulp more efficiently. Pretreatment of pulp with xylanase increases the whiteness and quality of the final paper product and reduces the amount of bleaching chemicals which must be used to bleach the pulp. This, in turn, decreases the amount of bleaching chemicals present in the effluent produced by such processes.
The most important chemical pulping process is the production of kraft pulp. For kraft pulp, following pulping, and prior to the treatment of pulp with xylanase, the pulp is exposed to a temperature of 55-70° C. and a highly alkaline pH (e.g. Nissen et al., 1992). A drawback of many commercially available wild-type xylanases is that these enzymes exhibit an acidic pH optimum and a temperature optimum of about 55° C. Therefore, in order to utilize xylanases effectively for bleaching applications, the pulp must be acidified to a pH approximating the optimal pH for the specific xylanase used. In addition, the hot pulp must be cooled to a temperature close to the optimal temperature for enzymatic activity of the selected xylanase. Decreasing pulp temperatures for xylanase treatment decreases the efficiency of the subsequent chemical bleaching. Acidification of pulp requires the use of large quantities of acids. Furthermore, the addition of acids leads to corrosion and lessens the lifetime of process equipment. Thus, xylanases optimally active at temperatures and pH conditions approximating the conditions of the pulp would be useful and beneficial in pulp manufacturing.
Xylanases which exhibit greater activity at higher temperatures could be used to treat pulp immediately following the pulping process, without the need to cool the pulp. Similarly, xylanases which exhibit greater activity at higher pH conditions would require less or no acid to neutralize the pulp. Xylanases with such properties would provide several advantages and substantial economic benefits within a variety of industrial processes.
Several approaches directed towards improving xylanase for use in pulp bleaching within the prior art include the isolation of thermostable xylanases from extreme thermophiles that grow at 80-100° C., such as Caldocellum saccharolyticum, Thermatoga maritima and Thermatoga sp. Strain FJSS-B.1 (Lüthi et al., 1990; Winterhalter et al., 1995; and Simpson et al., 1991). However, these thermostable xylanase enzymes are large, with molecular masses ranging from 35-120 kDa (320-1100 residues), and have a reduced ability to penetrate the pulp mass compared with other smaller xylanases which exhibit better accessibility to pulp fibers. In addition, some of the extremely thermophilic xylanases, such as Caldocellum saccharolyticum xylanase A, exhibit both xylanase and cellulase activities (Lüthi et al., 1990). This additional cellulolytic activity is undesirable for pulp bleaching due to its detrimental effect on cellulose, the bulk material in paper. Furthermore, hyper-thermostable xylanase enzymes, which function normally at extremely high temperatures, have low specific activities at temperatures in the range for optimal pulp bleaching (Simpson et al., 1991).
A number of xylanases have been modified by protein engineering to improve their properties for industrial applications. For instance, U.S. Pat. No. 5,405,769 (Campbell et al.) discloses the modification of Bacillus circulans xylanase (BcX) using site-directed mutagenesis to improve the thermostability of the enzyme. The site specific mutations include replacing two amino acids with cysteine residues to create intramolecular disulfide bonds. The mutations to create disulfide bonds include S179C (i.e., serine at position 179 replaced with cysteine) for an intermolecular crosslink between two xylanase molecules, and S100C/N148C and V98C/A152C for the creation of intramolecular crosslinks. These disulfide linkages contribute to the thermostability of the enzyme, and do not effect the thermophilicity or alkalophilicity of the enzyme. WO 00/29587 (Sung and Tolan) discloses the formation of the disulfide crosslinks, 110/154 and 108/158, in the fungal xylanase of Trichoderma reesei xylanase II (TrX or TrX II), corresponding to the 100/148 and 98/152 disulfide bonds of the BcX. As in the case of BcX, these crosslinks also increased the thermostability of TrX II, but do not have an effect on the thermophilicity or alkalophilicity of the enzyme.
U.S. Pat. No. 5,405,769 (supra) also discloses the mutation of specific residues in the N-terminus of the xylanase and these mutations were found to further improve the thermostability of the enzyme. In in vitro assays, the disulfide mutants showed thermostability at 62° C., an improvement of 7° C. over the native BcX xylanase enzyme. However, these thermostable disulfide mutants showed no gain in thermophilicity (Wakarchuck et al., 1994). Mutations T3G, (BcX xylanase amino acid numbering) D4Y(F) and N8Y(F), near the N-terminus of the BcX xylanase enzyme, provided thermostability to 57° C., an increase of 2° C. over the native BcX (U.S. Pat. No. 5,405,769). However, the use of these enzymes in industrial applications still requires cooling and acidification of pulp following pretreatment prior to enzyme addition. Therefore, further increases in thermostability, thermophilicity and pH optima are still required.
It is known in the art to modify Trichoderma reesei xylanase II (TrX II or TrX) to increase thermophilicity and alkalophilicity. For instance, U.S. Pat. No. 5,759,840 (Sung et al.) and U.S. Pat. No. 5,866,408 (Sung et al.) disclose mutations in the N-terminal region (residues 1-29) of TrX. Three mutations, at residues 10, 27 and 29 of TrX, were found to increase the enzymatic activity of the xylanase enzyme at elevated temperatures and alkaline pH conditions.
WO 01/92487 (Sung) discloses mutations S75A, L105R, N125A, I129E of TrX II, to produce a xylanase which maintains greater activity at higher temperature and pH. WO 03/046169 (Sung) also describes the application of multiple mutations to arginine residues (Y135R, H144R, Q161R) in order to increase the pH optimum of the TrX II. The mutation, Y118C, allowed the xylanase to maintain its optimal activity at higher temperature.
Turunen et al. (2002) describe the use of specific multiple arginines on the specific “Ser/Thr surface” of TrX II to increase the enzymatic activity at higher temperatures, but with decreased thermostability. It was also reported that another mutation, K58R, displayed slightly increased thermostability. However, this mutation in combination with other arginines showed a narrower range of effective pH.
Turunen et al. (2001) disclose mutations N11D, N38E, Q162H of TrX II with a complement of similar disulfide bonds (S110C/N154C) to improve the thermostability of the xylanase. However, these mutations, including N11D, also have an adverse effect on both the thermophilicity and the alkalophilicity of the xylanase, resulting in a decrease of enzymatic activity at higher temperatures and neutral-alkaline pH as compared to native TrX II.
There have been many attempts to stabilize proteins via the introduction of engineered disulfide bonds, with mixed results. Sowdhamini et al. (1989) describes a computational procedure called MODIP (Modeling of Disulfide bridges in Proteins) to aid in the design of proteins with disulfide bridges. By this method, a large number of sites for potential disulfide bond formation are usually predicted, with no way to foretell which are most likely to stabilize the protein. Dani et al. (2003) describe a refined version of this method to assist such selection. It predicted that a crucial requirement in any stabilizing disulfide bond is to enclose a loop of more than 25 amino acid residues between the two cysteines. A loop with less than 25 residues will offer little stabilization.
WO 00/29587 (Sung and Tolan) report the formation of two disulfide bonds in Trichoderma reesei xylanase II, one linking positions 110 and 154, and another linking positions 108 and 158 (both enclosed loops longer than 25 residues). Both disulfide bonds provide for enhanced thermostability of the enzyme, but do not enhance the thermophilicity.
Fenel et al. (2004) describe the formation of a disulfide bridge in TrX II through two mutations, T2C and T28C, which results in an increase in the temperature optimum and the thermostability of the enzyme without any change in the pH-dependent activity. The disulfide crosslink encloses a loop having a length of 26 amino acid residues between the two cysteine residues.
While the prior art discloses the modification of xylanases to alter various characteristics, the needs of current industrial processes require enzymes with increasingly robust activity. There is a need in the art for novel xylanases which exhibit increased enzymatic activity at elevated temperatures and pH conditions. Such enzymes would be adaptable to uses in various fields, for example the production of paper pulp and the washing of precision devices and semiconductors.